Electron transfer studies on cholesterol LB films assembled on thiophenol and 2-naphthalenethiol self-assembled monolayers

Journal of Colloid and Interface Science 315 (2007) 528–536 www.elsevier.com/locate/jcis

Electron transfer studies on cholesterol LB films assembled on thiophenol and 2-naphthalenethiol self-assembled monolayers Rakesh K. Pandey, K.A. Suresh, V. Lakshminarayanan ∗ Raman Research Institute, C.V. Raman Avenue, Bangalore 560080, India Received 18 April 2007; accepted 4 July 2007 Available online 27 July 2007

Abstract We have formed the cholesterol monolayer and multilayer LB films on the self-assembled monolayers of 2-naphthalenethiol (2-NT) and thiophenol (TP) and studied the electrochemical barrier properties of these composite films using cyclic voltammetry and electrochemical impedance spectroscopy. We have also characterized the cholesterol monolayer film using grazing angle FTIR, scanning tunneling microscopy (STM) and atomic force microscopy (AFM). Cholesterol has a long hydrophobic steroid chain, which makes it a suitable candidate to assemble on the hydrophobic surfaces. We find that the highly hydrophobic surface formed by the self-assembled monolayers (SAM) of 2-NT and TP act as effective platforms for the fabrication of cholesterol monolayer and multilayer films. The STM studies show that the cholesterol monolayer films on 2-NT form striped patterns with a separation of 1.0 nm between them. The area per cholesterol molecule is observed to be 0.64 nm2 with a tilt angle of about 28.96◦ from the surface normal. The electrochemical studies show a large increase in charge transfer resistance and lowering of interfacial capacitance due to the formation of the LB film of cholesterol. We have compared the behavior of this system with that of cholesterol monolayer and multilayers formed on the self-assembled monolayer of thiophenol. © 2007 Elsevier Inc. All rights reserved. Keywords: Cholesterol; Langmuir–Blodgett (LB) film; Self-assembled monolayer; 2-Napthalenethiol; Thiophenol

1. Introduction Cholesterol, a sterol and an amphiphilic molecule, is an essential component of cell membranes and some of the hormones and is known to alter several properties of lipid bilayers as it influences the permeability and fluidity of membranes [1–5]. We propose here a method of fabricating monolayer and multilayers films of cholesterol by a combination of self-assembled monolayer surface and Langmuir–Blodgett (LB) technique and use the composite film for the study of electron transfer properties of some redox active systems. Among several compounds that can form self-assembled monolayers (SAM), the chemisorption of thiols and disulfides on gold have attracted a great deal of attention especially due to their simplicity, ease of formation and for the opportunity they offer to tailor the surface properties by modifying the terminal func* Corresponding author. Fax: +91 80 23610492.

E-mail address: [email protected] (V. Lakshminarayanan). 0021-9797/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2007.07.044

tional groups [6,7]. The particularly attractive feature of the SAMs on a surface such as gold for the electrochemical studies is their remarkable stability over a wide potential window in aqueous media. There are very few reports in the literature on the studies of the composite self-assembled and Langmuir–Blodgett films. Recently Ashwell et al. have studied the rectification behavior in hybrid Au/SAM/LB device [8]. Yu et al. have studied the different bridges for interfacial electron transfer in azobenzene LB/SAM composite bilayers [9]. While cholesterol and its derivatives are known to form Langmuir monolayers at air– water interface [10–13] and LB films on substrates coated with hexamethyldisilazane [14], there are very few studies of these film using electrochemical and surface probe techniques [15]. Bin et al. have carried out the PM-IRRAS studies on the effect of cholesterol on the DMPC bilayer supported at Au electrode [16,17]. Yang et al. have reported the electrochemical and IRAS characterization of thiocholesterol SAM [18] and thiocholesterol, fatty acids mixed SAM [19]. To the best of our knowledge, there is no report in the literature on the study of electron

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transfer properties through the cholesterol monolayers and multilayer films formed on aromatic self-assembled monolayers. Such a study has the potential to improve our understanding of the biological processes across the cell membranes. The composite SAM and LB film can mimic the behavior of the soft membranes to provide a powerful model system for the study of transport of biomaterials across cell membranes. Ganesh et al. have recently reported the formation and characterization of the SAM of 2-naphthalenethiol (2-NT) using electrochemical techniques, STM and FTIR [20]. Due to its highly ordered structure, rich π electrons density and hydrophobic nature, this aromatic thiol monolayer can act as a substrate for the LB film formation and for electron transfer studies. It is known that the cholesterol molecules prefer hydrophobic surfaces and not hydrophilic surfaces to form LB films [14]. The 2-napthalenethiol SAM, with the hydrophobic aromatic group pointing upwards, therefore lends itself as an ideal platform for the deposition of cholesterol LB films to form the composite film. The aromatic thiol monolayer also facilitates electron transfer across the film, a useful property in the present studies involving composite films. For comparison, we have also formed the LB film of cholesterol on thiophenol SAM surface and characterized it using electrochemical techniques. Here, we report the results of our electron transfer and ion permeation studies on the cholesterol monolayers and multilayer films assembled on the SAMs of 2-naphthalenethiol (2-NT) and thiophenol (TP). We have used the redox system of [Fe(CN)6 ]3−/4− as a probe to evaluate the barrier properties of the system using electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The films have been characterized using STM, AFM and FTIR techniques. 2. Experimental 2.1. Chemicals All the chemicals used in this study were AR grade reagents. 2-NT (Aldrich), TP (Spectrochem), 5-cholest-3β-ol (cholesterol) (Aldrich), HPLC grade ethanol and chloroform (Merck). Millipore water of >18 M cm resistivity was used to prepare the aqueous solutions. Gold (∼100 nm thickness) on glass with chromium underlayers (2–5) nm was used as the substrate for SAM and LB film formation and characterization. The substrate was heated to 350 ◦ C during gold evaporation under a vacuum pressure of 2 × 10−5 mbar, a process that normally yields a deposit with predominantly Au (111) orientation. The evaporated gold samples with well defined area (about 0.2 cm2 ) were used as strips for electrochemical studies. A conventional three-electrode electrochemical cell was used for this study. A platinum foil of large surface area as the counter electrode and a saturated calomel electrode (SCE) as a reference electrode were used. The cell was cleaned thoroughly before each experiment and kept in a hot air oven at 100 ◦ C for at least 1 h for drying before the start of the experiment.

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Fig. 1. Surface pressure–area isotherm of cholesterol at the air–water interface. The deposition was carried out at a surface pressure of 30 mN/m.

2.2. Substrate pretreatment and thiol adsorption Evaporated Au strips were used for SAM formation and were pretreated with “piranha” solution (3:1 conc. H2 SO4 : H2 O2 ). Self-assembled monolayers were prepared by immersing the gold substrate in 1 mM 2-NT in ethanol and in 1 mM thiophenol in ethanol for 24 h. After the adsorption of thiol, the substrates were rinsed with ethanol, distilled water and finally with Millipore water and used for the LB deposition of cholesterol. STM, AFM, FTIR and electrochemical studies were also carried out on the modified substrate. 2.3. Deposition of cholesterol on SAMs Cholesterol solution of the concentration of 0.64 mg/ml was prepared in chloroform. The freshly prepared solution was spread using a microsyringe (Hamilton) on the Milli-Q Millipore water in a LB trough (NIMA 611M). The solvent was allowed to evaporate for 15 min before starting the compression. The surface pressure vs area per molecule experiments were carried out at room temperature (25 ◦ C). The target surface pressure was kept at 30 mN/m, which is well within the regime of monolayer (Fig. 1). This target surface pressure was optimized as the monolayer is found to be stable and transfer efficiency is also good. The steep region of the isotherm between 34 and 38 Å2 /molecule shows that the cholesterol molecules exhibit single condensed phase in this region. After attaining the target pressure, a duration of 15 min was allowed for the monolayer to attain the equilibrium. The monolayer of cholesterol was obtained from a single cycle of deposition while for the formation of two, three and four layers, repeated cycles of the respective number were performed. The speed of dipper was maintained at 10 and 5 mm/min for the downward and upward motions. 2.4. Electrochemical characterization Electrochemical studies were carried out by cyclic voltammetry and electrochemical impedance spectroscopy. The barrier properties of the SAM, cholesterol monolayer and multilayers have been evaluated by studying the electron transfer reaction using the potassium ferrocyanide/potassium ferricyanide as a redox probe. Cyclic voltammetry was performed in 1 mM

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potassium ferrocyanide with 1 M sodium fluoride as a supporting electrolyte at a potential range of −100 to 500 mV vs SCE. The impedance measurements were carried out by applying an ac voltage of 10 mV amplitude at the formal potential of the redox couple in a solution containing equal concentrations of both the oxidized and reduced forms of the redox couples (i.e., 1 mM potassium ferrocyanide and 1 mM potassium ferricyanide in 1 M NaF). A frequency range of 100 kHz to 100 mHz was used for impedance measurements. The interfacial capacitance measurements were carried out in a pure supporting electrolyte of 1 M NaF without any redox species. The capacitance values were obtained from the imaginary component of the impedance at high frequency regions where the value remains essentially constant. All the electrochemical studies were performed at 25 ◦ C. 2.5. Instrumentation Cyclic voltammetry was carried out using an EG&G potentiostat (model 263A) interfaced to a PC through a GPIB card. The potential ranges and scan rates used are shown in the respective diagrams. For electrochemical impedance studies, the potentiostat was used along with an EG&G 5210 lock in amplifier controlled by the Power Sine software. We have used a Pico plus (Molecular Imaging) AFM in tapping mode with an n-doped silicon tip. STM studies were carried out using a homemade STM in ultra low noise mode [21]. The STM was operated in constant current mode of 0.5 nA at a bias voltage of +100 mV. An electrochemically etched tungsten tip was used as the probe. STM and AFM images were obtained at 25 ◦ C in air. The images shown here are plane corrected and optimally Fourier filtered using scanning probe image processor (SPIP) software (Image Metrology, Denmark). To ensure that the images shown were representative of the monolayer morphology, multiple images were taken at different locations and scan ranges. The FTIR spectra were obtained using a FTIR 8400 model (Shimadzu) with a fixed 85◦ grazing angle attachment (FT-85; Thermo Spectra-Tech). 3. Results and discussion 3.1. Electrochemical characterization The electrochemical characterization of the cholesterol monolayer and multilayer films was carried out using cyclic voltammetry and electrochemical impedance spectroscopy in order to understand the electron transfer properties of the redox species across the film and also the ionic permeation through the film. 3.1.1. Cyclic voltammetry 2-Naphthalenethiol (2-NT) system Fig. 2 shows the cyclic voltammograms of (a) bare gold electrode, (b) 2-NT modified electrode, (c) monolayer of cholesterol modified SAM (denoted as Au/2-NT-SAM/1-ch) and (d) bilayer of cholesterol modified SAM (Au/2-NT-SAM/2-ch) in 1 mM potassium ferrocyanide with 1 M NaF as supporting electrolyte at potential scan rate of

Fig. 2. Cyclic voltammogram of (a) bare gold, (b) 2-NT-SAM, (c) 2-NT-SAM and cholesterol monolayer and (d) 2-NT-SAM and cholesterol bilayer in 1 mM potassium ferrocyanide solution with 1 M NaF as supporting electrolyte.

50 mV/s. It can be seen that bare gold electrode shows the reversible peaks for the redox couple, characteristic of a diffusion controlled reaction. On the other hand, the SAM of 2-NT modified electrode (Fig. 2b) shows an irreversible behavior with a large peak separation and lower peak currents. This indicates that the SAM of 2-NT considerably hinders the electron transfer process though not completely blocking it. This behavior is also typical of aromatic monolayer covered surfaces with poor blocking character towards the redox species [20]. It is generally known that aromatic thiols, due to their highly delocalized π electrons in the aromatic ring and extended conjugation of the molecules do not completely block the electron transfer reactions [22–28]. This property of the aromatic SAM is made use of in sensor and catalysis studies [25–27]. In contrast to 2-NT-SAM on gold, the CV of the cholesterol monolayer on 2-NT-SAM (Fig. 2c) shows almost complete blocking of the electron transfer reaction. This is due to the formation of well-organized and good blocking SAM-LB composite film on the surface of gold. The driving force for the organization of the cholesterol molecules on the surface of SAM is by hydrophobic interaction, which is very much stable and sustained in aqueous medium. It has been shown earlier that cholesterol forms a well-organized LB monolayer only on a hydrophobic surface and not on a hydrophilic surface [14]. This observation is supported by the fact that our efforts to form a stable LB film of cholesterol on hydrophilic surfaces of bare gold and 4-aminothiophenol modified gold were not successful as confirmed by the CV studies which showed very poor barrier properties towards potassium ferrocyanide redox reaction. Fig. 2d shows cyclic voltammogram of the sample after the formation of two layers of cholesterol on the 2-NT modified gold substrate. It can be seen that the cholesterol bilayer modified electrode has an excellent blocking towards the redox reaction, which is even better than that of the monolayer modified SAM electrode. This confirms that a stable film with excellent barrier properties for electron transfer has been achieved by forming a bilayer of cholesterol on the 2-NT-SAM. Thiophenol (TP) system Fig. 3 shows the cyclic voltammograms for the (a) bare gold, (b) TP-SAM, (c) monolayer of cholesterol modified thiophenol SAM (Au/TP-SAM/1-ch) and (d) bilayer of cholesterol modified SAM (Au/TP-SAM/2-ch).

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Fig. 3. Cyclic voltammogram of (a) bare gold, (b) TP-SAM, (c) TP-SAM and cholesterol monolayer and (d) TP-SAM and cholesterol bilayer in 1 mM potassium ferrocyanide solution with 1 M NaF as supporting electrolyte.

We find that the TP-SAM does not block the electron transfer process completely which is in agreement with the literature reports [29–32]. The composite film however, exhibits a remarkable improvement in the blocking property towards the ferrocyanide/ferricyanide redox couple. The order of the blocking ability is, Au < Au/TP-SAM < Au/TP-SAM/1-ch < Au/TPSAM/2-ch. However, we find that the electron transfer blocking ability of the monolayer and bilayer of cholesterol on thiophenol is less effective than in the case of 2-NT system. The CV studies discussed above are only qualitative indicator of the electron transfer process. In order to obtain quantitative information of the charge transfer resistance, we have carried out electrochemical impedance studies. 3.1.2. Electrochemical impedance analysis LB film of cholesterol on 2-NT system Fig. 4 shows the impedance plots of (a) Bare gold (b) SAM of 2-NT, (c) Au/2-

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NT-SAM/monolayer of cholesterol (1-ch) and (d) Au/2-NTSAM/bilayer of cholesterol (2-ch). The experiments were performed in equal concentrations of ferro/ferricyanide with 1 M NaF as the supporting electrolyte. It can be seen from Fig. 4a and the inset that the impedance plot for the bare gold electrode shows a very small semicircle at high frequency range and a straight line over a wide frequency window signifying a process that is fully under diffusion control. Fig. 4b shows the characteristic semicircle at higher frequencies representing the charge transfer process and a low frequency Warburg impedance for the 2-NT-SAM modified gold electrode. The diameter of the semicircle increases for the cholesterol monolayer and bilayer deposited on the SAM (Figs. 4c and 4d). The impedance plots were fitted to Randle’s equivalent circuit and the values of charge transfer resistance for each system were calculated. The equivalent circuit used for fitting the measured data are also shown in the respective Figs. 4a–4f. The typical Randle’s equivalent circuit is composed of a series uncompensated resistance Ru , a series combination of charge transfer resistance Rct and the Warburg impedance (W ), which are in parallel with the double layer capacitance Cdl or a constant phase element Q. The Warburg impedance, W is not considered wherever the low frequency Warburg region is absent in the impedance plot. The values obtained from fitting the impedance values for various samples are shown in Table 1. There is a remarkable increase in the charge transfer resistance (Rct ) value in Au/2-NT-SAM/1-ch system as compared to that of SAM modified electrode by a factor of about 30. We have calculated the surface coverage from the formula  (by assuming that the current is due to the presθ = 1 − Rct /Rct ence of the pinholes and defects within the monolayer) where

Fig. 4. Impedance Nyquist plots for (a) bare gold, (b) 2-NT-SAM, (c) 2-NT-SAM and cholesterol monolayer, (d) 2-NT-SAM and cholesterol bilayer, (e) 2-NT-SAM and cholesterol trilayer and (f) 2-NT-SAM and cholesterol tetralayer in 1 mM potassium ferrocyanide/potassium ferricyanide solution with 1 M NaF as supporting electrolyte.

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Table 1 Charge transfer resistance (Rct ), constant phase element (Q) and its exponent (n) for ferrocyanide–ferricyanide redox reaction from the equivalent circuit fitting of electrochemical impedance spectroscopy for 2-NT system Electrode type

Rct (Ohm cm2 )

Q (S sn cm−2 )

n (0 < n < 1)

Bare gold SAM of 2-NT Cholesterol monolayer on 2-NT-SAM Cholesterol bilayer on 2-NT-SAM Cholesterol trilayer on 2-NT-SAM Cholesterol tetralayer on 2-NT-SAM

7.60 [RC(RW)] 592.1 [RC(RW)] 19.57K [R(QR)]

– – 2.4 × 10−6

– – 0.97

2.406M [R(QR)]

1.27 × 10−6

0.96

786.0K [R(QR)]

1.4 × 10−6

0.97

1.47M [R(QR)]

1.3 × 10−6

0.97

Rct is the charge transfer resistance of bare gold electrode and  is the charge transfer resistance of the corresponding modiRct fied electrode. For the cholesterol monolayer the surface coverage is calculated to be 99.94%. The bilayer of cholesterol shows very high charge transfer resistance, which is more than two orders of magnitude higher compared to the Au/2-NT-SAM/1-ch system. The surface coverage in this case is calculated to be 99.99%. This extremely high resistance is attributed to the hydrophobic nature of cholesterol that is exposed to the solution, which inhibits the diffusion of ions through the film and towards the electrode surface. Cholesterol monolayer has the hydrophilic nature because of –OH group being in the upright direction, the bilayer system is hydrophobic which is formed by two cycles of deposition. We have extended the bilayer by two additional cholesterol layers in order to evaluate the integrity and stability of the films. We find that trilayer, which is formed by three cycles of deposition interestingly shows less charge transfer resistance value than the Au/2-NT-SAM/2-ch system (Fig. 4e). This again is due to the fact that the trilayer has the hydrophilic nature since three cycles of deposition creates a surface with the –OH group of cholesterol exposed to the solution. The hydrophilic character of the film helps the charged ions in solution to permeate the film. Moreover, the presence of multiple layers increases the defects in the film, which opens the path for the diffusing ions to permeate through the film. Interestingly, the deposition of fourth layer increases the resistance of the film, which is higher than Au/2-NT-SAM/3-ch system (Fig. 4f) though not so high as for the bilayer system. This is because, after the formation of fourth layer, the surface again acquires hydrophobic character, which makes it difficult for the ions to permeate through the film. However the charge transfer resistance is less than Au/2-NT-SAM/2-ch system (Fig. 4b). This implies that due to the weak adsorption, the formation of further layers above the bilayer introduces more voids and defects by disorienting the cholesterol molecules. This facilitates the permeability of ions from the solution and consequently decreases the charge transfer resistance compared to the more compact Au/2-NT-SAM/2-ch system. We have also carried out interfacial capacitance studies using electrochemical impedance spectroscopy (EIS). The capac-

Table 2 Capacitance values obtained from electrochemical impedance spectroscopy in 1 M NaF supporting electrolyte for 2-NT system Electrode type

Measured capacitance (µF/cm2 )

Calculated capacitance (µF/cm2 )

SAM of 2-NT Cholesterol monolayer on 2-NT-SAM Cholesterol bilayer on 2-NT-SAM Cholesterol trilayer on 2-NT-SAM Cholesterol tetralayer on 2-NT-SAM

2.60 1.76 1.10 1.13 1.14

2.55 0.92 0.55 0.40 0.31

itance of the electrical double layer precisely describes the adsorption properties and is being used widely in the study of thin films on metal surfaces [33,34]. We have earlier shown that the interfacial capacitance can be precisely measured in pure supporting electrolyte and by carefully selecting the high frequency region of the impedance plot where the capacitance essentially remains constant [34]. The experiments were performed in 1 M NaF solution without any redox species. The capacitance values measured in this case are shown in Table 2. The values show that the additional layers beyond the bilayer do not change the measured capacitance values significantly. This shows that the small sodium and fluoride ions can penetrate through the cholesterol film of third and fourth layers comparatively easily thereby effectively restricting the dielectric thickness to the bilayer over SAM. Table 2 shows that the measured capacitance value for 2-NTSAM as 2.6 µF/cm2 , which corresponds to the expected value of the thickness based on the height of 2-NT (0.8 nm) molecules. This indicates that a compact film of 2-NT is formed on the surface of gold. The measured capacitance value of 1.76 µF/cm2 for the cholesterol monolayer on 2-NT-SAM is higher than the expected value of 0.92 µF/cm2 by assuming the total thickness of the composite film to be 2.4 nm and dielectric constant ε = 2.5. Since the cholesterol film acts as a dielectric film over the 2-NT-SAM, the total measured capacitance of 1.76 µF/cm2 is the effective series capacitance of both the layers. From this value, the cholesterol film capacitance alone can be calculated to be 5.5 µF/cm2 . This value is rather large when compared to the theoretically expected capacitance value of 1.38 µF/cm2 for a compact film. This suggests that the cholesterol layer over 2NT-SAM has several defects through which the ions can quite easily access the electrode surface. However, the charge transfer resistance for the ferro/ferricyanide has increased considerably for this system when compared to 2-NT-SAM. This implies that in spite of the large number of defects, the redox reaction is considerably inhibited. Based on this observation, we propose that the defect sites in the 2-NT-SAM have been filled by cholesterol, which therefore effectively blocks the electron transfer reaction. The cholesterol molecules, which fill the defect sites, may also be anchored by the hydrophobic interaction with neighboring 2-NT molecules. Besides, there can be a tilt of the cholesterol molecules over the surface of the self-assembled monolayer, which can decrease the thickness and increase the capacitance. This model is schematically depicted in Fig. 5. We

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Fig. 5. Schematic representation of different layers of cholesterol on 2-NT-SAM (a) monolayer of cholesterol, (b) bilayer of cholesterol, (c) trilayer of cholesterol and (d) tetralayer of cholesterol. Cholesterol molecule is depicted showing the hydrophobic and hydrophilic ends in the figure.

Fig. 6. Impedance Nyquist plots for (a) bare gold, (b) 2-TP-SAM, (c) 2-TP-SAM and cholesterol monolayer, (d) 2-TP-SAM and cholesterol bilayer, (e) 2-TP-SAM and cholesterol trilayer and (f) 2-TP-SAM and cholesterol tetralayer in 1 mM potassium ferrocyanide/potassium ferricyanide solution with 1 M NaF as supporting electrolyte.

also suggest that ‘plugging’ of the defect sites may lead to the formation of more defects in the first cholesterol monolayer on 2-NT-SAM. The presence of large number of defect sites formed thereby effectively increases the interfacial capacitance of the cholesterol film to 5.5 µF/cm2 . The addition of a second cholesterol layer on top of the first one further lowers the interfacial capacitance to 1.10 µF/cm2 . The charge transfer resistance Rct has considerably increased to 2.4 M cm2 which confirms the formation of a compact bilayer of cholesterol on the 2-NT self-assembled monolayer on gold.

There is however, little variation in capacitance beyond the bilayer. It is therefore concluded that the size and number of the defects increase in the higher layers, which facilitate the permeation of ions closer to the surface till up to a certain distance. Thiophenol–cholesterol system We compare here the electron transfer properties of 2-NT system with that of thiophenol system. Fig. 6 shows the impedance curves for thiophenol system. Table 3 shows the charge transfer resistance values for the thiophenol system increasing systematically from SAM of TP right

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Table 3 Charge transfer resistance (Rct ) and constant phase element (Q) for ferrocyanide–ferricyanide redox reaction obtained by electrochemical impedance spectroscopy for TP system Electrode type

Rct (Ohm cm2 )

Q (S sn cm−2 )

n (0 < n < 1)

Bare gold SAM of TP Cholesterol monolayer on TP-SAM Cholesterol bilayer on TP-SAM Cholesterol trilayer on TP-SAM Cholesterol tetralayer on TP-SAM

7.60 [RC(RW)] 462.5 [RQ(RW)] 1.1K [RQ(RW)]

– 1.6 × 10−5 6.6 × 10−6

– 0.93 0.92

2.34K [RQ(RW)]

2.8 × 10−6

0.93

76.0K [R(Q(RW))]

1.5 × 10−6

0.98

308.0K [R(QR)]

1.6 × 10−6

0.97

Table 4 Capacitance values obtained from electrochemical impedance spectroscopy in 1 M NaF solution for TP system Electrode type

Measured capacitance (µF/cm2 )

Calculated capacitance (µF/cm2 )

SAM of TP Monolayer of cholesterol on TP-SAM Bilayer of cholesterol on TP-SAM Trilayer of cholesterol on TP-SAM Tetralayer of cholesterol on TP-SAM

3.96 1.33 0.93 1.27 1.31

2.50 1.10 0.62 0.43 0.33

up to the four layers of cholesterol. However, the measured charge transfer resistance values are less than the corresponding values of 2-NT system. We also find that the interfacial capacitance values for Au/TP-SAM/1-ch, Au/TP-SAM/3-ch and Au/TP-SAM/4-ch are almost the same. The thiophenol self-assembled monolayer shows a capacitance value of 3.96 µF/cm2 which is quite higher than the theoretically expected value of 2.5 µF/cm2 . This may indicate a large tilt in the molecules forming the monolayer as has been suggested earlier by Sabatani et al. [29]. In this system, the capacitance of the cholesterol monolayer alone on TP-SAM is calculated to be 2.0 µF/cm2 . This value of capacitance is somewhat larger when compared to the theoretically calculated capacitance of 1.38 µF/cm2 assuming a vertical orientation of the cholesterol molecules. The reason for this increase could be due to the presence of defects as well the tilt in the cholesterol arrangement on the surface of TP-SAM. Significantly, the Rct value for the ferrocyanide–ferricyanide redox system is not quite large as it is in the case of cholesterol layer on 2-NT. The rather low value of Rct is also consistent with the cyclic voltammetry results, which shows very poor blocking of the redox reaction both for the TP-SAM and the cholesterol on TP-SAM. There is however marked decrease in the capacitance of Au/TP-SAM/2-ch system from that of the Au/TP-SAM system. Table 4 shows the capacitance values for thiophenol– cholesterol system. The measured capacitance for the Au/TPSAM/1-ch is smaller than that of the Au/2-NT-SAM/1-ch, which indicates that the thickness of the film formed by the

former system is more. This is rather surprising since the thiophenol is a shorter molecule and therefore TP-SAM is expected to be shorter than 2-NT-SAM. The measured capacitance value in the case of Au/TP/1-ch is close to the expected value based on the thickness of the film (0.4 nm). There is no change in the capacitance beyond a bilayer in the case of thiophenol system. This may indicate that after the formation of bilayer, the system becomes more permeable for sodium and fluoride ions. These ions approach closer to the surface, which decreases the effective separation between the electrode surface and the solution. In other words, the third and fourth layers of cholesterol molecules do not function as effective dielectric separators. 3.2. Scanning probe analysis of cholesterol monolayer on 2-NT-SAM 3.2.1. AFM studies We have carried out tapping mode AFM studies on bare gold surface, 2-NT-SAM and the cholesterol monolayer film over the SAM system (Fig. 7). Fig. 7a shows the bare gold substrate, which has crystalline domains typical of the vacuum, deposited and annealed surface at this scan range. The AFM image of 2-NT-SAM on Au essentially follows the crystalline domains seen on the bare gold surface (Fig. 7b). However, the AFM image of cholesterol monolayer formed on the 2NT-SAM (Fig. 7c) shows several elongated domains, which were absent in 2NTSAM on gold. The elongated features present in cholesterol LB film can be attributed to the formation of the cholesterol monolayer along the direction of dipping of the substrate in the LB trough [35,36]. 3.2.2. STM studies STM imaging has been carried out in constant current mode using +100 mV sample bias voltage and 1 nA tunneling current to confirm the organization of cholesterol molecules and to obtain information on the structural and orientational aspects of the cholesterol molecules arranged on the 2-NT-SAM. The structure of 2-NT-SAM on Au(III) has already been investigated using STM and reported by our group [20]. √ It was shown that it forms a well aligned ( 3 × 3)R 30◦ over layer structure on Au(III) surface. Fig. 8a shows the monolayer of cholesterol molecules on the SAM of 2-NT on evaporated gold surface. It can be seen that cholesterol molecules arrange themselves in rows forming a striped pattern. Fig. 8b shows several bright features in the image. The bright features can be attributed to higher electron density polar –OH groups oriented in the upright direction. The calculated area occupied by individual cholesterol molecule from STM images is 0.64 nm2 . The tilt angle can be measured from the ratio of the true area of the molecule and area per molecule from STM image [37]. This is found to be 28.96◦ from the surface normal. The STM studies confirm the formation of a well-aligned monolayer of cholesterol molecules on 2-NT-SAM. This is also in accordance with our electrochemical and AFM results.

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Fig. 7. Tapping mode AFM images of (a) bare gold (b) 2-NT-SAM on Au surface and (c) cholesterol monolayer on 2-NT-SAM/Au.

Fig. 8. Constant current STM images of (a) cholesterol monolayer on 2-NT-SAM showing individual cholesterol molecules arranged in rows (b) 3-d view of cholesterol monolayer on 2-NT-SAM showing individual cholesterol molecules. The lines drawn on the image show 0.5 nm along the rows and 1 nm along the columns. Tunneling current = 1 nA; Bias voltage = +100 mV (substrate).

Fig. 9. Grazing angle FTIR spectra of (a) cholesterol film on 2-NT-SAM and (b) cholesterol film on TP-SAM.

3.3. Grazing angle FTIR analysis We have carried out grazing angle FTIR spectroscopy on the Au/2-NT-SAM/LB system to confirm the organization of cholesterol on SAMs. Figs. 9a and 9b are the FTIR spectra of the Au/2-NT/cholesterol and Au/TP/cholesterol respectively. We have observed the typical cholesterol peaks in both cases that show the presence of cholesterol. The peaks are representative of the different modes of vibrations present in the choles-

terol. In 2-NT system we have well defined peaks at 2833, 2886 and 2912 cm−1 which correspond to the CH2 C–H symmetric stretch, CH3 C–H symmetric stretch and CHC–H asymmetric stretch respectively. Also we have peaks at 2941 and 2991 cm−1 showing CH2 C–H asymmetric stretch and CH3 C–H asymmetric stretch, respectively. The transmittance peaks are observed in case of thiophenol system. Here the different peaks at 2841, 2889 and 2918 cm−1 correspond to the CH2 C–H symmetric stretch, CH3 C–H symmetric stretch and CHC–H asymmetric

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stretch, respectively. The peaks at 2938 and 2967 cm−1 correspond to CH2 C–H asymmetric stretch and CH3 C–H asymmetric stretch, respectively. The grazing angel FTIR spectra confirm the coverage of cholesterol monolayer on the 2-NT-SAM. 4. Conclusion In this work, we have studied the electron transfer and ion permeation properties through the cholesterol monolayers and multilayer films formed on self-assembled monolayers of thiophenol and 2-NT on Au substrate. The molecular films of cholesterol have also been characterized using STM, AFM and Grazing angle FTIR studies. We find that the charge transfer resistance for the potassium ferrocyanide–ferricyanide redox system has increased remarkably by the deposition of cholesterol layers on the SAM. The charge transfer resistance and interfacial capacitance values depend upon the exposed part of the cholesterol molecules towards electrolyte and also the structure of the monolayer. The modified electrodes show almost complete blocking of the electron transfer reaction. Interfacial capacitance studies with EIS confirm the formation of LB films onto the SAM. The STM and AFM studies of the cholesterol monolayer show that it forms a good, compact and well-ordered film on 2-napthalenethiol SAM. The area per molecule is measured to be 0.64 nm2 for cholesterol molecule with a tilt angle of about 28.96◦ from the surface normal as obtained from STM studies. We feel that these studies are important in understanding the structure, orientation, ordering, electron transfer and ion permeation properties of biologically important cholesterol molecules. Acknowledgments We thank Mr. H. Subramonyam Ram for the preparation of evaporated gold specimens. References [1] M. Sugahara, M. Uragami, X. Yan, S.L. Regen, J. Am. Chem. Soc. 123 (2001) 7939. [2] D. Needham, R.S. Nunn, Biophys. J. 58 (1990) 997. [3] F.A. Nezil, M. Bloom, Biophys. J. 61 (1992) 1176. [4] M. Doxastakis, A.K. Sum, J.J. De Pablo, J. Phys. Chem. B 109 (2005) 24173. [5] K. Tu, M.L. Klein, D.J. Tobias, Biophys. J. 75 (1998) 2147.

[6] H.O. Finklea, in: R.A. Meyers (Ed.), Encyclopedia of Analytical Chemistry, Wiley, Chichester, 2000; (b) A. Ulman, An Introduction to Ultrathin Organic Films from Langmuir– Blodgett to Self-Assembly, Academic Press, San Diego, CA, 1991; (c) G. Roberts, Langmuir–Blodgett Films, Plenum Press, New York, 1990. [7] A. Ulman, Chem. Rev. 96 (1996) 1533. [8] G.J. Ashwell, M. Sujka, A. Green, Faraday Discuss. 131 (2006) 23. [9] H.Z. Yu, N. Xia, J. Zhang, Z.F. Liu, J. Electroanal. Chem. 448 (1998) 119. [10] P. Viswanath, K.A. Suresh, Phys. Rev. E 67 (2003) 061604. [11] P. Viswanath, K.A. Suresh, J. Phys. Chem. B 108 (2004) 9198. [12] R.S. Abendan, J.A. Swift, Langmuir 18 (2002) 4847. [13] S. Lafont, H. Rapaport, G.J. Somjen, A. Renault, P.B. Howes, K. Kjaer, J. Als-Nielsen, L. Leiserowitz, M. Lahav, J. Phys. Chem. B 102 (1998) 761. [14] R.K. Gupta, K.A. Suresh, Eur. Phys. J. E 14 (2004) 35. [15] B.W. Gregory, R.A. Dluhy, L.A. Bottomley, J. Phys. Chem. 98 (1994) 1010. [16] X. Bin, S.L. Horswell, J. Lipkowski, Biophys. J. 89 (2005) 592. [17] X. Bin, J. Lipkowski, J. Phys. Chem. B 110 (2006) 26430. [18] Z.P. Yang, I. Engquist, J.M. Kauffmann, B. Liedberg, Langmuir 12 (1996) 1704. [19] Z.P. Yang, I. Engquist, B. Liedberg, J.M. Kauffmann, J. Electroanal. Chem. 430 (1997) 189. [20] V. Ganesh, V. Lakshminarayanan, J. Phys. Chem. B 109 (2005) 16372. [21] M. Jayadevaiah, V. Lakshminarayanan, Meas. Sci. Technol. 15 (2004) N35. [22] N. Krings, H.-H. Strehblow, J. Kohnert, H.-D. Martin, Electrochim. Acta 49 (2003) 167. [23] M.A. Rampi, G.M. Whitesides, Chem. Phys. 281 (2002) 373. [24] Q. Jin, J.A. Rodriguez, C.Z. Li, Y. Darici, N.J. Tao, Surf. Sci. 425 (1999) 101. [25] C. Retna Raj, Takeo Ohsaka, J. Electroanal. Chem. 540 (2003) 69. [26] T. Sawaguchi, F. Mizutani, S. Yoshimoto, I. Taniguchi, Electrochim. Acta 45 (2000) 2861. [27] Y. Yang, S.B. Khoo, Sens. Actuators B 97 (2004) 221. [28] R.R. Kolega, J.B. Schlenoff, Langmuir 14 (1998) 5469. [29] E. Sabatani, J. Boulakia-Cohen, M. Bruening, I. Rubinstein, Langmuir 9 (1993) 2974. [30] T. Sawaguchi, F. Mizutani, S. Yoshimoto, I. Taniguchi, Electrochim. Acta 45 (2000) 2861. [31] Y.-T. Tao, C.-C. Wu, J.Y. Eu, W.L. Lin, K.-C. Wu, C.-H. Chen, Langmuir 13 (1997) 4018. [32] S. Frey, V. Stadler, K. Heister, W. Eck, M. Zharnikov, M. Grunze, B. Zeysing, A. Terfort, Langmuir 17 (2001) 2408. [33] H.O. Finklea, in: A.J. Bard, I. Rubinstein (Eds.), Electroanalytical Chemistry, in: A Series of Advances, vol. 19, Dekker, New York, 1996, p. 166. [34] R. Subramanian, V. Lakshminaryanan, Electrochim. Acta 45 (2000) 4501. [35] S.J. Tans, L.J. Geerligs, C. Dekker, J. Wu, G. Wegner, J. Vac. Sci. Technol. B 15 (1997) 586. [36] W. Zhu, N. Minami, S. Kazaoui, Y. Kim, J. Mater. Chem. 13 (2003) 2196. [37] P. Jiang, A. Nion, A. Marchenko, L. Piot, D. Fichou, J. Am. Chem. Soc. 128 (2006) 12390.